Listeria monocytogenes is a foodborne bacterial pathogen with the ability to grow at refrigeration temperatures, and is thus a psychrotolerant organism. It is an intracellular pathogen and its molecular mechanisms of pathogenesis have been studied extensively. In order to grow at low temperatures psychrotolerant organisms must adjust their fatty acid compositions in order to maintain membrane fluidity. Under normal conditions almost the entire complement (more than 90%) of L. monocytogenes fatty acids are branched-chain fatty acids, in contrast to other Gram-positive bacteria that typically have about 40% straight-chain fatty acids. Fatty acid anteiso C15:0 increases in low-temperature grown bacteria by a combination of fatty acid shortening and branching switching from iso to anteiso fatty acids, and plays a key role in increasing membrane fluidity. Study of branched-chain fatty acid-deficient mutants has shown they are significantly impaired in low temperature growth, tolerance of various stresses, and virulence. This indicates that the physical structure and optimum function of the Listeria membrane is dependent on a high content of branched-chain fatty acids, and deficiency in these has major impacts on the physiology and virulence of the organism. Mutant and precursor feeding studies have revealed the existence of a novel but undefined pathway of fatty acid biosynthesis from short straight- and branched-chain carboxylic acid precursors, in addition to the normal pathway from branched-chain amino acids. We propose to study the mechanism of branched-chain fatty acid switching, define the pathway of fatty acid biosynthesis from carboxylic acids, and manipulate the membrane fatty acid composition, and hence fluidity, in an extreme fashion and examine its impact on the physiology and pathogenesis of the organism. The major determinant of fatty acid branching switching appears to reside in the temperature- responsive catalytic properties of the enzyme -keto acyl carrier protein synthase III (FabH) that carries out the first condensation reaction in the fatty aid biosynthesis pathway. In specific aim I we will combine mutagenesis, functional and in silico modeling, protein biochemistry and kinetic analyses to probe the mechanisms underlying temperature-dependent variation of substrate specificities of FabH. Short- and branched-chain carboxylic acids by pass the normal fatty acid biosynthetic pathway in both branched-chain fatty acid-deficient mutants and wild-type organisms. In specific aim 2 we will clone, overexpress the proteins encoded by buk, butyrate kinase, and ptb, phosphotransbutyrylase, and attempt to show that they constitute a novel pathway for production of the CoA derivatives of carboxylic acid fatty acid precursors. L. monocytogenes has the ability of use branched-chain C6 carboxylic acid as precursors of unnatural even-numbered branched-chain fatty acids. In specific aim 3 we will use precursor feeding experiments to produce bacteria of low, normal and high fluidity membranes through manipulation of their fatty acid composition. These cells will be used to study the impact of membrane physical structure and fluidity on low temperature tolerance, various aspects of cell physiology, and bacterial pathogenesis. Realization of these objectives will increase our understanding of growth at low temperatures and the impact of membrane structure on physiology and pathogenesis. It is hoped that novel ways of controlling the growth of Listeria at low temperatures will emanate from the work. PUBLIC HEALTH RELEVANCE: Listeria monocytogenes is a foodborne pathogen with a high fatality rate and its ability to grow in foods at refrigeration temperatures is a critical factor i this. The proposal focuses on the role of branched-chain fatty acids in membrane physical structure, psychrotolerance, physiology, and pathogenicity. Novel methods for controlling the growth of the organism are expected to be developed as a result of the work.